1. Field of the Invention
This invention relates to a non-cariogenic composition and drink for preventing or at least inhibiting human dental caries induced by Streptococcus mutans and other cariogenic bacilli.
2. Description of the Prior Art
Dental caries is a disease of teeth of humans and animals, which is induced by the action of cariogenic oral bacilli. Among them, Streptococcus mutans is believed to be most important. It has, hitherto, been disclosed, for example, in Bergy's Manual of Determinative Bacteriology, page 504 (1974) that a relationship exists between dental caries and Streptococcus mutans, it being suggested that S. mutans is a similar microorganism to S. salivarius, even though S. mutans has not yet been extensively studied and compared with S. salivarius. Nowadays, however, S. mutans which produces dextran-like polysaccharides (hereinafter referred to as DPS) from sucrose, is clearly distinguished from S. salivarius which produces fructans from sucrose. Strains of S. mutans adher to the surface of the teeth of humans and animals and form dental plaque which is solid and adhesive. The major portion of S. mutans in the oral cavity resides in the plaque and produces lactic acid which directly breaks down the teeth. Even though other oral bacilli also produce lactic acid, the lactic acid produced by S. mutans is not released but is accumulated on the surface of the teeth. Thus, for example, British Patent 1,375,866 discloses that the formation of pre-carious dental plaque is a prerequisite for the development of dental caries and that glucosyltransferease is an important factor in the development of dental caries. It is said that the main constituents of insoluble and water-soluble DPS produced by S. mutans are high molecular weight polymers of glucose comprising respectively .alpha.(1-3) and .beta.(1-6) bondings (Guggenheim, Internat. Dent. J., 20:675-678, 1980).
There are known various other cariogenic microorganisms other than S. mutans. For example, it is well known that certain bacilli conventionally used for the preparation of yogurt, cheese and various other foodstuffs containing lactic acid-producing bacilli have cariogenic potential. For example British Patent 1,375,866 discloses that Lactobacillus acidophilus, L. casei and Streptococcus faecalis are well known aetiological factors. Oshima also reported that L. casei and L. helvetius used for yogurts commercially available in Japan induced dental caries (The Japanese Journal of Pedodontics, 16, 1. 161-169, 1978 in Japanese version). Even though the cariogenic potential of other cariogenic bacilli is not higher than that of S. mutans, for example, they intrude into dental caries already induced by S. mutans and reside there. In such a case, for example, lactic acid and acetic acid produced by them do harm to the structure of the teeth.
It is known to inhibit (as hereinbefore defined) dental caries induced by S. mutans by using dental vaccine. Thus, for example, said British Patent 1,375,866 discloses a dental vaccine comprising as antigen the whole cells of S. mutans Strain SSC having the same characteristics as the characteristics of S. mutans NTCT 10449 which is a well-recognized cariogenic wild type strain of S. mutans. However, it is said that the usu of the whole cells (especially, the living whole cells) of cariogenic strains of S. mutans as antigen, can give rise to various undesired side effects such as allergic reaction and cross reaction with the heart muscle antigen, when administered to animals.
The use of bovine milk containing anti-S. mutans antibodies for inhibiting dental caries induced by S. mutans is also known (British Patent 1,505,513). However, the known bovine milk antibodies may not be used in practice since the resultant antibodies cannot inhibit S. mutans NCTC 10449, a well-recognized cariogenic strain of Human type S. mutans.
Apart from the known immunological methods, U.S. Pat. No. 4,133,875 to Hillman (1979) discloses to control human dental caries induced by cariogenic strains of S. mutans by the use of non-virulent analogs of cariogenic S. mutans. Such a method is based upon the socalled bacterial interference which appears, in general, to involve a competitive and/or antibiotic reaction of the non-virulent strains viz. the effector strain on its pathogenic counterpart and that such a strain must be (a) non-virulent as itself and (b) able to successfully compete with its pathogenic counterpart (cf. U.S. Pat. No. 4,133,875, column 3, lines 5-15).
Hillman discloses two effector strains, Streptococcus mutans JH140 (ATCC 31341) and JH145 (ATCC 31377), both isolated from streptomycin-resistant S. mutans BHT-2 (str.), followed by mutagensis. The biological characteristics of two effectors strains are, for example, as follows:
(a) inability to produce a detectable amount of lactic acid when incubated in the presence of glucose;
(b) relatively better adhesion to hydroxypate than the parent strain; and
(c) higher productivity of plaque than the parent strain when incubated in the presence of sucrose.
However, S. mutans JH140 and JH145 did not give good result when used in practice for controlling human dental caries and, by way of example, the following problems arose:
(1) S. mutans is highly mutative and is classified into certain strain types. For example, Bratthall classified S. mutans into 5 serotypes (a to e) in view of the immunological specificities of the polysaccharides contained in the cell wall (Odont. Revy., 20:143, 1970; ibid. 20:13-27, 1970; and Perch et al, Microbiol. Scand Section B. 82:357, 1974).
Makoto Sato, one of the coinventors of this invention, has classified S. mutans into Human I (HI), Human II (HII) and Rat (R) types with respect to the specific antigens of S. mutans, which respectively correspond to said "`c`, `e` and `f`", "`d` and `g`" and "`a` and `b`" serotypes. Sato has reported:
(a) 93.9% of S. mutans of human origin were Human I type and the rest was Human II type;
(b) all strains of rat origin were Rat type;
(c) no S. mutans was isolated from mice and guinea pigs; and
(d) all strains originating from hamsters and monkeys were Human I type. (Journal of Dental Health. Vol. 28, No. 2, pages 99-123, 1978 in Japanese version).
However, BHT-2, the parent strain of JH140 and JH145 are the well known strain of Rat type S. mutans which has bever been isolated from humans and which differs from Human type strains. What will happen by administering Rat type strain to humans is not yet entirely clear.
(2) JH140 and JH145 form a large amount of plaque and have a higher ability to adher than the parent strain. However, it is common knowledge in the art that the formation of plaque and adhesion to the surface of the teeth result in the induction of dental caries.
3. Objects of the Invention
As is well known, S. mutans is highly mutative and has been classified into certain types. However, the competitive relationship between different strain types has not yet been completely clarified. In order to solve this question, we have produced many mutant strains by treating various wild type strains of S. mutans with mutagents such as, for example, nitrogen mustard, nitrosoguanidine or iradiation of ultraviolet rays etc. and have studied their characteristics such as, for example, colonial characteristics, biological characteristics, cariogenic potentials and the like. As a result, we have classified the mutational phases of S. mutans into the following 3 phases in disregard of their strain types:
(1) Phase I
"Normal" phase viz. the mutational phase of cariogenic wild type strains present in the oral cavity of humans and animals. When cultured on a sucrose-containing agar plate medium such as, for example, TYC agar plate medium (Stoppelaar et al, Archs. Oral Biol., 12, 1199-1201, 1967), solid colonies covered with a large amount of insoluble DPS are formed. Each colony has an irregular and raised margin and its central area is rough. There are minor morphological difference between respective colonies. The plaque formed is abundant, solid and adhesive and adhers to the tube wall and on the surface of the teeth. The productivity of lactic acid is high, and the cariogenic potential is highest of all 3 phases.
(2) Phase II
Essentially unstable phase, obtainable by treating Phase I viz. wild type cariogenic strain by mutagent. Also this phase occur sometines by subculturing Phase I. There is a mutational variability between Phases I and II. When cultured on a sucrose-containing agar plate medium, round colonies like water-drops are formed, which are bright and somewhat mucoid. There are considerable morphological difference between respective colonies. The productivity of lactic acid and cariogenicity are weaker than those of Phase I.
(3) Phase III
Obtainable by treating Phase I with mutagent. Various characteristics of this phase are entirely different from the characteristics of other phases. For example, when cultured on a sucrose-containing agar plate medium, the colonies formed are round, bright, opaque smooth and large. In the test tube, the productivity of lactic acid is low, and other acids than lactic acid are substantially reduced. The ability to produce insoluble and water-soluble DPS is substantially reduced.
With respect to the competitive relationship between Phases I and III, it has been found that Phase III strains are capable of inhibiting the growth of Phase I strains in coexistence with them in vitro and in vivo. Where Phase III is introduced into a culture or into the oral cavity of animals where the plaque has been formed by the action of Phase I strains, Phase III intrudes into the plaque to "reside" there so that Phase I strains are outwardly destroyed and gradually disappeare owing to the loss of residence. Phase III strains also die owing to the loss of their support medium and inability to form new plaque. Where Phase I is introduced into a test tube or oral cavity already occupied by Phase III strains, Phase I strains die.
Various characteristics of Phase III are genetically stable. For example, no reversion to the parent strains was observed nor was there any observable generation of other mutant strain by subculturing on various media such as, for example, TYC agar plate media and no such effects were observed when continuously administered to hamsters, both subculturing and continuous administration being effected over an extended period of time (more than 6-12 months).
Based upon the above-mentioned discovery, Tsurumizu and Hashimoto, the coinventors of this invention, proposed a dental vaccine, comprising as active ingredient the living cells of Streptocococcus mutans Attenuated Strain K-III (FERM BP 316) which they produced by treating a cariogenic strain of S. mutans isolated from the oral cavity of a human with mutagent (disclosed in Japanese Patent Application 47970/82 filed on Mar. 25, 1982 and laid open to public inspection on Sep. 29, 1983). Said K-III strain exhibits good results when used for inhibiting human dental caries induced by S. mutans. However, with respect to the commercial value of Phase III mutant strains, for example, the following problems arose: (a) very complicated procedure is required for obtaining official license for the commercial production of vaccines, and moreover (b) in many countries of the world, it is, as a rule, extremely difficult or impossible to use the viable cells of mutant strains obtained by treating virullent microorganisms with mutagents, for example, as additive to foodstuffs.
In order to overcome such difficulties and also eliminate the need for further processing which would otherwise be necessary to enable direct and safe administration into the oral cavity of humans, we have collected very many strains from the oral floras of more than 1000 humans and have compared their characteristics with the charcteristics of Phase III strains which we have induced from various cariogenic strains of S. mutans. As a result, it has unexpectedly been found that strains of S. mutans having analogous characteristics to the characteristics of Phase III mutant strains (hereinafter referred to as Phase III-like strains) are living in the oral cavity of minor humans, particularly, children and infants. It should be noted in this reagrd that no or little, if any, dental caries is observed in the oral cavity of the human hosts of Phase III-like strains. The reason why such unique strains exist preferably in the oral floras of children and infants is not yet clear. We have succeeded to isolate and purely culture various Phase III-like strains. It has been confirmed that Phase III-like strains are capable of effectively competing with cariogenic strains of S. mutans and moreover capable of inhibiting various other cariogenic oral bacilli in the oral cavity of humans with good result.
An object of the present invention is to provide a non-cariogenic composition for inhibiting (as hereinbefore defined) human dental caries, which may be used simply and safely with good results.